First class: Basic skills in a molecular biology lab

Transcription

1 First class: Basic skills in a molecular biology lab a) Prepare digest with BamHI or BglII, for the following DNAs: FUGW, genomic DNA, FUCEdW. b) Prepare 1% agarose gel c) Precipitate genomic DNA d) Run digest e) Take pictures

2 Basic Procedure: Liquid Handling CAL 5 Ex 20 o C Figure by MIT OCW.

3 Basic Procedure: Liquid Handling : µl. Use large tip. 200: µl. Use small tip. 20: 2-20 µl. Use small tip.

4 Basic Procedure: Centrifuge Have connector always outside Balance tubes. Complete stop before opening.

5 Plasmid What is plasmid? What is the difference of plasmid and usual DNA?

6 Plasmid What is plasmid? Small circular DNA. Replicated inside of host bacteria. Can carry extra gene such as drug resistance gene. Can carry foreign DNA. What is the difference of plasmid and usual or chromosomal DNA? Size Chromosomal DNA carries most (>%99.99) of necessary gene for bacteria Bacteria can live without plasmid DNA but not without chromosomal DNA.

7 Restriction Enzymes The critical component of DNA recombinant technology Why it is called restriction enzyme? How often do you see these sites in DNA? BamHI BglII Why restriction enzyme is useful?

8 Application of restriction enzyme

9 Separation of DNA fragment What is the relationship of migration distance and molecular weight? Include your analysis in the report.

10 Visualization of DNA in agarose gel Ethidium bromide (EtBr): Possible carcinogen.

11 Protocols Preparation of the digest Cut 3 μ of each type of DNA with BamHI or BglII. For BamHI use buffer 2 (blue) For BglII use buffer 3 (red tube) Cut the following: FUGW with BamHI FUGW with BglI FUGW with no enzyme FUGW with both BamHI and BglII (in buffer 2) FUCed with BglII FUCed with no enzyme Genomic DNA with BglII (per tube) 3 µl of DNA 2 µl of the appropriate buffer (red or blue) 14 µl of water 1 µl of BSA (bovine serum albumin) Why do you think we have BSA? 0.5 µl of enzyme (BamHI or BglII) Mix by gently tapping. Spin for 3 seconds Incubate in 37C waterbath for at least 40 minutes (while waiting, start preparing the gel )

12 Protocols Preparation of the agarose gel Measure 100 ml of TAE buffer in measuring syrinder Pour 50 ml of TAE buffer in a 200 ml bottle Measure 1 gram of agarose in a weigh boat Add agarose to bottle and swirl thoroughly Loosen cap and put in microwave for 2 minutes at low power (4) Make sure that contents do not boil and spill over Add the rest of TAE to the bottle and mix well by swirling Add 5 µl of ethidium bromide to liquid in bottle Swirl (without making bubbles) Cast get with two 12 tooth combs (one on top row and one in bottom) Add agarose into cast and let solidify Wait 15 minutes (while you wait, precipitate the genomic DNA as explained below) Once is solid, remove comb, turn the cast 90 degrees, and add approx. 400 ml of TAE buffer, until the liquid starts to cover the gel

13 Protocols Precipitation of DNA (this is separate experiment to be done while waiting for digestion and electrophoresis) Into a 1.5 ml eppendorf tube, add: 40 µl of genomic DNA 5 µl of sodium acetate 3 M 100 µl of 100% ethanol Mix well by shaking Spin in centrifuge at maximun speed for 5 minutes Discard supernatant in waste bucket Add 1 ml of 70% ethanol Discard supernatant in waste bucket (make sure that your remove all you can by inverting tube on a piece of paper and gently tap the tube to eliminate all remaining drops) Air dry tube for 5 minutes Add 10 µl of water to bottom of tube Tap bottom of tube to ensure mixing of water and pellet Spin down for 3 seconds Do you see something on the bottom?

14 Protocols Running of DNA digests and precipitation Load 4 µl of 1Kb DNA ladder in first well Mix 2 µl of loading buffer (LB tube) with each of the digests that you prepared before Load 23 µl of each of the digest+loading buffer in each well Add 3 µl of loading buffer to the precipitated genomic DNA, and load into a well The loading order should be: 1kb ladder-fugw (BaMHI)- FUGW (BglII)-FUGW (BamHI and BglII)-FUGW (no enzyme)- genomic DNA (BglII) -FUCedW (BglII)- FUCed (no enzyme)-precipitated genomic DNA (plus empty wells) Once everything is loaded, set voltage to 150 volts and start running the gel Run gel for 15 minutes Stop gel and take image with UV light

15 Report What is the relationship between migration distance and molecular weight? How can you calculate unknown DNA size? How many BamHI and BglII sites are there? How are they arranged? Draw a restriction enzyme map of FUWG What is the smeary pattern? (No introduction, method, etc needed for today. Just the result of you analysis and a map of the plasmid. Write about possibilities.)